Technical Field
The present disclosure relates generally to the highly sensitive quantification of the relative representation of adaptive immune cells having a particular T cell receptor (TCR) or immunoglobulin (Ig) encoding gene rearrangement (e.g., clonotype), in samples obtained from a subject prior to and/or following therapeutic treatment. Determination of the relative frequencies of such clonotypes can be used to diagnose certain lymphoid hematologic malignancies and other disorders. Also, determination of the relative frequencies of such clonotypes following therapeutic treatment provides exquisitely sensitive detection of minimal residual disease (MRD).
Description of the Related Art
The adaptive immune system protects higher organisms against infections and other pathological events that may be attributable to foreign substances, using adaptive immune receptors, the antigen-specific recognition proteins that are expressed by hematopoietic cells of the lymphoid lineage and that are capable of distinguishing self from non-self molecules in the host. These lymphocytes may be found in the circulation and tissues of a host, and their recirculation between blood and the lymphatics has been described, including their extravasation via lymph node high endothelial venules, as well as at sites of infection, inflammation, tissue injury and other clinical insults. (See, e.g., Stein et al., 2005 Immunol. 116:1-12; DeNucci et al., 2009 Crit. Rev. Immunol. 29:87-109; Marelli-Berg et al., 2010 Immunol. 130:158; Ward et al., 2009 Biochem. J. 418:13; Gonzalez et al., 2011 Ann. Rev. Immunol. 29:215; Kehrl et al., 2009 Curr. Top. Microb. Immunol. 334:107; Steinmetz et al., 2009 Front. Biosci. (Schol. Ed.) 1:13.)
Accordingly, the dynamic nature of movement by lymphocytes throughout a host organism is reflected in changes in the qualitative (e.g., antigen-specificity of the clonally expressed adaptive immune receptor (immunoglobulin or T cell receptor), T cell versus B cell, T helper (Th) cell versus T regulatory (Treg) cell, effector T cell versus memory T cell, etc.) and quantitative distribution of lymphocytes among tissues, as a function of changes in host immune status.
The adaptive immune system employs several strategies to generate a repertoire of T- and B-cell antigen receptors with sufficient diversity to recognize the universe of potential pathogens. B lymphocytes mature to express antibodies (immunoglobulins, Igs or Igs, also referred to as B cell receptors, BCR) that occur as heterodimers of a heavy (H) a light (L) chain polypeptide, while T lymphocytes express heterodimeric T cell receptors (TCR). The ability of T cells to recognize the universe of antigens associated with various cancers or infectious organisms is conferred by its T cell antigen receptor (TCR), which is a heterodimer comprising an α (alpha) chain and a β (beta) chain, or a γ (gamma) and a δ (delta) chain. The proteins which make up these chains are encoded by DNA, which employs a unique mechanism for generating the tremendous diversity of the TCR. This multi-subunit immune recognition receptor associates with the CD3 complex and binds to peptides presented by the major histocompatibility complex (MHC) class I and II proteins on the surface of antigen-presenting cells (APCs). Binding of TCR to the antigenic peptide on the APC is a central event in T cell activation, which occurs at an immunological synapse at the point of contact between the T cell and the APC.
Each TCR peptide contains variable complementarity determining regions (CDRs), as well as framework regions (FRs) and a constant region. The sequence diversity of αβ T cells is largely determined by the amino acid sequence of the third complementarity-determining region (CDR3) loops of the α and β chain variable domains, which diversity is a result of recombination between variable (Vβ), diversity (Dβ), and joining (Jβ) gene segments in the β chain locus, and between analogous Vα and Jα gene segments in the α chain locus, respectively. The existence of multiple such gene segments in the TCR α and β chain loci allows for a large number of distinct CDR3 sequences to be encoded. CDR3 sequence diversity is further increased by independent addition and deletion of nucleotides at the Vβ-Dβ, Dβ-Jβ, and Vα-Jα junctions during the process of TCR gene rearrangement. In this respect, immunocompetence is reflected in the diversity of TCRs.
The γδ TCR is distinctive from the αβ TCR in that it encodes a receptor that interacts closely with the innate immune system. TCRγδ, is expressed early in development, has specialized anatomical distribution, has unique pathogen and small-molecule specificities, and has a broad spectrum of innate and adaptive cellular interactions. A biased pattern of TCRγ V and J segment expression is established early in ontogeny as the restricted subsets of TCRγδ cells populate the mouth, skin, gut, vagina, and lungs prenatally. Consequently, the diverse TCRγ repertoire in adult tissues is the result of extensive peripheral expansion following stimulation by environmental exposure to pathogens and toxic molecules.
Igs (BCR) expressed by B cells are proteins consisting of four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), forming an H2L2 structure. Each pair of H and L chains contains a hypervariable domain, consisting of a VL and a VH region, and a constant domain. The H chains of Igs are of several types, μ, δ, γ, α, and β. The diversity of Igs within an individual is mainly determined by the hypervariable domain. Similar to the TCR, the V domain of Ig H chains is created by the combinatorial joining of the VH, DH, and JH gene segments. Hypervariable domain sequence diversity is further increased by independent addition and deletion of nucleotides at the VH-DH, DH-JH, and VH-JH junctions during the process of Ig gene rearrangement. In this respect, immunocompetence is reflected in the diversity of Igs.
Quantitative characterization of adaptive immune cells based on the presence in such cells of functionally rearranged Ig and TCR encoding genes that direct productive expression of adaptive immune receptors has been achieved using biological samples from which adaptive immune cells can be readily isolated in significant numbers, such as blood, lymph or other biological fluids. In these samples, adaptive immune cells occur as particles in fluid suspension. See, e.g., US 2010/0330571; see also, e.g., Murphy, Janeway's Immunobiology (8th Ed.), 2011 Garland Science, NY, Appendix I, pp. 717-762.
Acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive, immature, malignant T-cell neoplasm that affects both adult and pediatric patients. While there has been significant progress in treating these patients with improvements in achieving durable responses, it remains clear that a subset of these patients are inadequately treated and frequently present with disease relapse, while others may be over-treated due to an inability to sufficiently individualize clinical treatment. Several studies have confirmed the importance of assessing the potential presence of minimal residual disease (MRD) following a treatment regimen, to aid in predicting clinical outcomes of patients (1-3). For example, patients who demonstrate an early response to therapy, and who fail to exhibit sustained achievement of MRD, fare significantly better than those who do not (3).
Similarly, acute B-cell lymphoblastic leukemia/lymphoma (B-ALL) is an aggressive immature malignant B-cell neoplasm that affects adult and pediatric patients. While significant progress has been made to increase the number of patients who achieve durable long-term remission, a subset of patients relapses. As in T-ALL, multiple studies have confirmed that the presence and frequency of minimal residual disease are both important prognostic markers in B-ALL. In addition, several of these studies also support the use of these data to inform and individualize therapy (e.g., Yamaji et al., 2010 Pediatr. Blood Canc. 55(7):1287-95; Bhojwani et al., 2009 Clin. Lymphoma Myeloma 9 (Suppl. 3):5222-30.
Current clinical strategies for assessment of minimal residual disease include multi-parameteric flow cytometry (mpFC) and quantitative PCR-based methods using patient-specific primers (4, 5). mpFC typically permits detection of cells potentially responsible for recurrent/persistent disease with a sensitivity of on the order of 10−4 to 10−6 nucleated cells. However, interpretation of these data is operator- and laboratory-dependent, and consequently limited by poor standardization. Furthermore, variable expression of leukemic antigens in the post-therapy setting confounds MRD detection by mpFC (6).
By comparison, molecular-based methods for detection of minimal residual disease can achieve relatively increased sensitivity, on the order of 10−5 to 10−6 cells (7, 8). However, the previous configurations of these molecular assays, principally real-time quantitative PCR-based (RT-qPCR) assays using patient-specific primers that target adaptive immune receptor (e.g., T-cell receptor (TCR) or immunoglobulin (Ig)) variable region junctional sequences, or patient-specific translocations, are complex and challenging to implement in a uniform matter (7). For instance, these approaches require the production and use of individualized, patient-specific oligonucleotide probes for each patient, which is laborious, costly and time-consuming, and incompatible with the timeframe in which clinical decisions must be made.
High-throughput sequencing (HTS) is an emerging technology that can provide insight into the complexity of the adaptive immune response through the analysis of lymphoid receptor gene rearrangement (9). Studies using this technology have challenged understanding in the art of the extent of lymphocyte diversity occurring within, and shared by, individuals (9, 10), and have provided mechanistic insight into the early molecular genetic events critical for the T-cell lineage maturation (11). Recently, high-throughput sequencing of lymphoid cell adaptive immune receptor genes has been used to monitor lymphocyte diversity after adoptive immunotherapy with chimeric antigen receptor-modified T cells for the treatment of chemotherapy-refractory chronic lymphocytic leukemia (12). Separately, high-throughput sequencing of lymphoid cell adaptive immune receptor genes has been used for monitoring disease in B lymphoproliferative disorders (13). HTS has exhibited the ability to identify rare T cell clones (one T cell in 100,000) with high accuracy and reproducibility (14).
Clearly there is a need for improved sensitivity and specificity in the diagnosis of lymphoid hematological malignancies and other conditions that are reflected in the heterogeneity and relative frequencies of occurrence of particular unique adaptive immune receptors, and in the ability to detect minimal residual disease (MRD). The presently described embodiments address these needs and provide other related advantages.